Dimerization Motif Research Articles

Decoding the impact of neighboring amino acids on ESI-MS intensity output through deep learning

Peptide-level quantification using mass spectrometry (MS) is no trivial task as the physicochemical properties affect both response and detectability. The specific amino acid (AA) sequence affects these properties, however the connection between sequence and intensity output remains poorly understood. In this work, we explore combinations of amino acid pairs (i.e., dimer motifs) to determine a potential relationship between the local amino acid environment and MS1 intensity. For this purpose, a deep learning (DL) model, consisting of an encoder-decoder with an attention mechanism, was built. The attention mechanism allowed to identify the most relevant motifs. Specific patterns were consistently observed where a bulky/aromatic and hydrophobic AA followed by a cationic AA as well as consecutive bulky/aromatic and hydrophobic AAs were found important for the prediction of the MS1 intensity. Correlating attention weights to mean MS1 intensities revealed that some important motifs, particularly containing Trp, His, and Cys, were linked with low responding peptides whereas motifs containing Lys and most bulky hydrophobic AAs were often associated with high responding peptides. Moreover, Asn-Gly was associated with low response. The model predicts MS1 response with a mean average percentage error of ∼11 % and a Pearson correlation coefficient of ∼0.64. While dimer representation of peptide sequences did not improve predictive capacity compared to single AA representation in earlier work, this work adds valuable insight for a better understanding of peptide response in MS analysis. SignificanceMass spectrometry is not inherently quantitative, and the response of a compound relies not only on its concentration but also on the molecular composition. For mass spectrometry-based analysis of peptides, such as in bottom-up proteomics, this directly implies that the response cannot be used directly to quantify individual peptides. Moreover, the dependency of the response on the amino acid sequence of individual peptides remains poorly understood. Using a deep learning model based on a recurrent neural network with an attention mechanism, we here investigate how the presence of dimer motifs within a peptide affects the MS1 response through the analysis of intended equimolar peptide pools comprising almost 200,000 unique peptides in total. Not only do we identify certain dimer classes and specific dimers that substantially affect the MS1 response, but the model is also able to predict peptide intensity with low error rates within the independent test subset. The findings not only improve our understanding of the link between sequence and response for peptides but also highlight the potential of utilizing deep learning for developing methods allowing for absolute, label-free peptide quantification.

Abstract A085: Engineered novel bioactive Nectin-4 dimer protein significantly enhances the binding of TIGIT

Abstract The goals of this study are to: (a) create a novel soluble bioactive Nectin-4 dimer molecule mimicking its native dimer quaternary structure on the cell surface and (b) evaluate the recombinant Nectin-4 dimer binding potency to its receptor vs the monomer form, so that the dimer can be used as an immunogen and an antigen to increase the potential of cancer therapeutic antibody and vaccine discovery when targeting quaternary structural epitopes. Nectin-4 is a cell adhesion molecule belonging to the nectin family. It's involved in cell-cell junctions and plays a role in cancer progression by influencing cell adhesion, migration, and proliferation. Nectin-4 is overexpressed on various types of tumor cells including breast, lung, ovarian and pancreatic cancers. Nectin-4 has been recently identified as a novel ligand of T cell immunoglobulin and ITIM domain (TIGIT) and interaction between Nectin-4 and TIGIT inhibits NK cell cytotoxicity thus suppressing immune responses, allowing cancer cells to evade immune surveillance. Nectin-4 and Nectin-1 interact in a heterophilic manner, however in the context of cancer, altered expression of Nectin-4 and Nectin-1 can contribute to tumor progression. Nectin-4 is a type 1 single transmembrane protein, and can form homodimers on the cell surface. While therapeutics such as Enfortumab Vedotin, an antibody-drug conjugate, binds Nectin-4 on the surface of cancer cells, mimicking the Nectin-4 dimer quaternary structure can present many more critical opportunities for therapeutic antibody and vaccine discovery. To better mimic the native dimer conformation and quaternary structure, we designed a novel soluble Nectin-4 dimer with 3 extracellular domains fused to a dimer motif at the C-terminus. The Nectin-4 dimer protein was expressed and purified from HEK293 cells and confirmed by SDS-PAGE and Western blot analyses. We characterized the Nectin-4 dimer protein binding potency to TIGIT and Nectin-1 by ELISA. Very interestingly, the results demonstrated that the Nectin-4 dimer binding potency to TIGIT increased dramatically as measured by ELISA, compared to the Nectin-4 monomer protein. Conversely, the Nectin-4 dimer binding to Nectin-1 monomer was significantly decreased, compared to Nectin-4 monomer, as expected. We also studied Nectin-4 dimer immunogenicity using a Nectin-4 dimer DNA immunization to induce potent antibody responses in mice. In summary, these findings imply that the Nectin-4 dimer protein is bioactive and in the desirable conformation, resulting in an increased binding potency to its receptor TIGIT, reduced binding to Nectin-1 (as expected), and eliciting strong antibody responses in mice. This novel Nectin-4 dimer protein can be a very useful molecule for cancer research, therapeutic antibody and vaccine discovery. The Nectin-4 dimer protein can be used to: (i) elicit antibody responses against the dimer quaternary structure, (ii) screen antibodies targeting more conformational epitopes, and (iii) study the Nectin-4 and TIGIT ligand/receptor interactions. Citation Format: Christofer Walsh, Timothy Smith, Mikhail Rashkovskii, Richard Parent, Jean Qiu, Shixia Wang. Engineered novel bioactive Nectin-4 dimer protein significantly enhances the binding of TIGIT [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A085.

Hydrogen Bond Strengthens Acceptor Group: The Curious Case of the C-H···O=C Bond.

An H-bond involves the sharing of a hydrogen atom between an electronegative atom to which it is covalently bound (the donor) and another electronegative atom serving as an acceptor. Such bonds represent a critically important geometrical force in biological macromolecules and, as such, have been characterized extensively. H-bond formation invariably leads to a weakening within the acceptor moiety due to the pulling exerted by the donor hydrogen. This phenomenon can be compared to a spring connecting two masses; pulling one mass stretches the spring, similarly affecting the bond between the two masses. Herein, we describe the opposite phenomenon when investigating the energetics of the C-H···O=C bond. This bond underpins the most prevalent protein transmembrane dimerization motif (GxxxG) in which a glycine Cα-H on one helix forms a hydrogen bond with a carbonyl in a nearby helix. We use isotope-edited FT-IR spectroscopy and corroborating computational approaches to demonstrate a surprising strengthening of the acceptor C=O bond upon binding with the glycine Cα-H. We show that electronic factors associated with the Cα-H bond strengthen the C=O oscillator by increasing the s-character of the σ-bond, lowering the hyperconjugative disruption of the π-bond. In addition, a reduction of the acceptor C=O bond's polarity is observed upon the formation of the C-H···O=C bond. Our findings challenge the conventional understanding of H-bond dynamics and provide new insights into the structural stability of inter-helical protein interactions.

Two New Polymorphs of Meso-Chlorinated BODIPY Dyes

New polymorphs of (2-(chloro(pyrrol-2-ylidene)methyl)pyrrole)-difluoro-borane (1) and (2-(chloro(4-ethyl-3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)pyrrole)-4-ethyl-3,5-dimethyl-1H-pyrrolato)-difluoro-borane (2) were identified using single crystal X-ray diffraction. The new polymorph of 1 crystallises in the triclinic, P-1 space group with an asymmetric unit comprising two crystallographically-independent molecules (Z′ = 2). The differences between this structure and that of the known polymorph of 1 are attributed to the formation of two distinct dimer motifs in each polymorph and the packing between these dimer units. The new polymorph structure of 2, exhibits a similar asymmetric unit to the known form, comprising 3 molecules (Zʹ = 3). The two structures differ in the orientation of the molecules within the trimeric asymmetric unit and the manner in which these trimers pack along the crystallographic [010] direction.Graphical The triclinic polymorph of 8-chloro BODIPY exhibits an asymmetric unit with two crystallographically-independent molecules.

Polyoxometalate-Dicarboxylate Hybrid Dimers with {SiW10Cr2} Nodes: Syntheses, Structures, and Mass Spectrometric Characterization.

The current study describes a new family of hybrid dimers constructed from secondary building blocks based on a dichromium-substituted silicodecatungstate, {SiW10Cr2}, and flexible dicarboxylate ligands of varied lengths. All five polyoxometalate-organic hybrid compounds exhibit an entropically favored, cyclic dimer motif that contains just two {SiW10Cr2} clusters─the minimum to form a closed loop, linked by two aliphatic dicarboxylates, as revealed by single-crystal X-ray diffraction. Due to the kinetic inertness of such Cr3+-based hybrid dimers, ESI-MS has become a particularly useful technique for the characterization of these paramagnetic, NMR-unfriendly systems.

Domain Swapping in Abiotic Foldamers

AbstractFoldamer sequences that adopt tertiary helix‐turn‐helix folds mediated by helix‐helix hydrogen bonding in organic solvents have been previously reported. In an attempt to create genuine abiotic quaternary structures, i.e. assemblies of tertiary structures, new sequences were prepared that possess additional hydrogen bond donors at positions that may promote an association between the tertiary folds. However, a solid state structure and extensive solution state investigations by Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) show that, instead of forming a quaternary structure, the tertiary folds assemble into stable domain‐swapped dimer motifs. Domain swapping entails a complete reorganization of the arrays of hydrogen bonds and changes in relative helix orientation and handedness that can all be rationalized.

Domain Swapping in Abiotic Foldamers.

Foldamer sequences that adopt tertiary helix-turn-helix folds mediated by helix-helix hydrogen bonding in organic solvents have been previously reported. In an attempt to create genuine abiotic quaternary structures, i.e. assemblies of tertiary structures, new sequences were prepared that possess additional hydrogen bond donors at positions that may promote an association between the tertiary folds. However, a solid state structure and extensive solution state investigations by Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) show that, instead of forming a quaternary structure, the tertiary folds assemble into stable domain-swapped dimer motifs. Domain swapping entails a complete reorganization of the arrays of hydrogen bonds and changes in relative helix orientation and handedness that can all be rationalized.

The apo-acyl coenzyme A binding protein of Leishmania major forms a unique ‘AXXA’ motif mediated dimer

Acyl-Coenzyme A binding domain containing proteins (ACBDs) are ubiquitous in nearly all eukaryotes. They can exist as a free protein, or a domain of a large, multidomain, multifunctional protein. Besides modularity, ACBDs also display multiplicity. The same organism may have multiple ACBDs, differing in sequence and organization. By virtue of this diversity, ACBDs perform functions ranging from transport, synthesis, trafficking, signal transduction, transcription, and gene regulation. In plants and some microorganisms, these ACBDs are designated ACBPs (acyl-CoA binding proteins). The simplest ACBD/ACBP is a small, ∼10 kDa, soluble protein, comprising the acyl-CoA binding (ACB) domain. Most of these small ACBDs exist as monomers, while a few show a tendency to oligomerize. In sync with those studies, we report the crystal structure of two ACBDs from Leishmania major, named ACBP103, and ACBP96 based on the number of residues present. Interestingly, ACBP103 crystallized as a monomer and a dimer under different crystallization conditions. Careful examination of the dimer disclosed an exposed ‘AXXA’ motif in the helix I of the two ACBP103 monomers, aligned in a head-to-tail arrangement in the dimer. Glutaraldehyde cross-linking studies confirm that apo-ACBP103 can self-associate in solution. Isothermal titration calorimetry studies further show that ACBP103 can bind ligands ranging from C8 – to C20-CoA, and the data could be best fit to a ‘two sets of sites’/sequential binding site model. Taken together, our studies show that Leishmania major ACBP103 can self-associate in the apo-form through a unique dimerization motif, an interaction that may play an important role in its function.

Structural determinants for membrane binding of the EGFR juxtamembrane domain.

Overactivation of the epidermal growth factor receptor (EGFR) is critical for the development of multiple cancers. Previous studies have shown that the cell membrane is a key regulator of EGFR kinase activity through its interaction with the EGFR juxtamembrane domain (JM). However, the lipid recognition specificity of EGFR-JM and its interaction details remain unclear. Using lipid strip and liposome pulldown assays, we showed that EGFR-JM could specifically interact with PI(4,5)P2-or phosphatidylserine-containing membranes. We further characterized the JM-membrane interaction using NMR-titration-based chemical shift perturbation and paramagnetic relaxation enhancement analyses, and found that residues I649 - L659 comprised the membrane-binding site. Furthermore, the membrane-binding region contains the predicted dimerization motif of JM, 655LRRLL659, suggesting that membrane binding may affect JM dimerization and, therefore, regulate kinase activation.

Crystallographic and theoretical analysis of a spirohydantoin derivative: 3-(4‑tert-butylbenzoyl)-1,3-diazaspiro[4.5]decane-2,4‑dione

Providing structural information on drug-like compounds is a significant step in efforts to develop innovative drugs with balanced solubility, cell permeability and target binding. In this paper, a new cyclohexane-5-spirohydantoin tethered with a 4‑tert-butylbenzoyl group was synthesized and its crystal structure was determined using the single crystal X-ray diffraction. The Hirshfeld surface analysis was used to gain a preliminary insight into the proportion and nature of the intermolecular interactions in the crystal structure, while their quantitative description is further presented in terms of a systematic analysis of dimeric motifs representing different recognition modes. A combination of N–H∙∙∙O and C(sp3)–H∙∙∙O hydrogen bonds, C(sp3)–H∙∙∙π, lone-pair∙∙∙π and π∙∙∙π stacking interactions links the molecules into a bilayer having two carbonyl O atoms exposed to the exterior. Further stacking of the bilayers occurs through C(sp3)–H∙∙∙O hydrogen bonds involving these O atoms as acceptors, C(sp3)–H∙∙∙π and π∙∙∙π stacking interactions between the phenyl groups. The molecular electrostatic potential surface map reveals that the carbonyl O atoms of the hydantoin ring and the phenyl ring are electrophilic centers, while the N1–H1 group is a nucleophilic center. The calculated HOMO and LUMO energies were used to semiquantitatively estimate the global reactivity descriptors suggesting the soft nature of the molecule.

Crystal structure, hydrogen bonding interactions, Hirshfeld surfaces, energy frameworks, and DFT calculation of Diethyl 3-(4-substitutedbenzoyl)indolizine-1,2-dicarboxylates

The diethyl 3-(4-substitutedbenzoyl)indolizine-1,2-dicarboxylates have been synthesized by reacting the electron-deficient diethyl but-2-ynedioate with 1-(2-(4-substitutedphenyl)-2-oxoethyl)pyridin-1-ium bromide, the intermediates obtained from the reaction of pyridine and 4-substituted phenacyl bromides. The synthesized compounds were investigated by FT-IR, NMR (1H and 13C), LC-MS, and elemental analysis spectroscopic methods along with PXRD. The molecular structures were determined using the single crystal X-ray diffraction technique which revealed that 5a and 5c crystallize in triclinic space group P1¯. The crystal structures of 5a and 5c preferred the C-H···O hydrogen-bonded dimeric ring motifs, resembling those observed in the earlier reported 5b. Furthermore, various computational analyzes such as Hirshfeld surface, Energy framework, and MEP analysis predicted the interaction site and inter-molecular interactions in these molecules. The FMOs analysis was carried out to obtain global reactivity parameters for the synthesized compounds (5a-c). The NCI and QTAIM studies revealed the nature as well as qualitative and quantitative description of intra and inter-molecular hydrogen bonding interactions present in these molecules and their crystal packing.

Abstract 1344 Role of GXXXG Dimerization Motif and Lipid-Protein Interactions in the Functional Modulation of Glutamate Carboxypeptidase II

Abstract 1344 Role of GXXXG Dimerization Motif and Lipid-Protein Interactions in the Functional Modulation of Glutamate Carboxypeptidase II

Functionality optimization for effective singlet fission coupling screening in the full-dimensional molecular and intermolecular coordinate space.

In computational chemistry, accurately predicting molecular configurations that exhibit specific properties remains a critical challenge. Its intricacies become especially evident in the study of molecular aggregates, where the light-induced functionality is tied to highly structure-dependent electronic couplings between molecules. Here, we present an efficient strategy for the targeted screening of the structural space employing a "functionality optimization" technique, in which a chosen descriptor, constrained by the ground state energy expression, is optimized. The chosen algorithmic differentiation (AD) framework allows one to automatically obtain gradients without its tedious implementation. We demonstrate the effectiveness of the approach by identifying perylene bisimide (PBI) dimer motifs with enhanced effective SF coupling. Our findings reveal that certain structural modifications of the PBI monomer, such as helical twisting and bending as well as slipped-rotated packing arrangements, can significantly increase the effective SF coupling.

Soluble meso and deuteroporphyrin analogs of the malaria pigment hematin anhydride

Two soluble heme analogs of the insoluble malaria pigment hematin anhydride (HA, or β-hematin), [Fe(III)(protoporphyrin)]2, with either mesoporphyrin (MHA) or deuteroporphyrin (DHA) are characterized by elemental analysis, SEM, IR spectroscopy, electronic spectroscopy, paramagnetic 1H NMR spectroscopy and solution magnetic susceptibility. While prior single crystal and X-ray powder diffraction results indicate all three have a common propionate linked dimer motif, there is considerable solid state variation in the conformation. This is associated with enhanced solubility of MHA and DHA. As with HA, DHA undergoes thermally promoted reversible hydration/dehydration in the solid state. Solution 1H NMR studies of DHA suggest a high spin dimeric structure with the porphyrin methyls distributed between two isomers which are also present in the solid state. These soluble iron(III)porphyrin dimers allow for the first direct solution studies by NMR and UV–Vis spectroscopies of these key species. Taken together the results illustrate the importance and utility of varying the substituents on the periphery of the porphyrin for studying heme aggregation and malaria pigment formation.

Exploring protein-protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching.

Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.

Synthesis and structural characterization of novel di-substituted pyridine-2,6-dicarboxylic acid hydrazide oxo-quinazoline derivative and Cu(II) pincer complex

Crystal and molecular structures of novel pyridine-2,6-dicarboxylic acid (pdc) hydrazide derivative (H2L) dimethanol solvate (H2L = N,N'-bis(2-methyl-4-oxoquinazolin-3(4H)-yl)pyridine-2,6-dicarboxamide) and its copper(II) complex were determined by the single-crystal X-ray diffraction. In the crystal structure, these non-planar molecules form specific cavities that are populated by methanol molecules (held by OH⋯O and NH⋯O hydrogen bonds). Additional stabilization is achieved through a series of offset face-to-face π⋯π interactions between aromatic moieties. The compound is the first structurally described pdc hydrazide derivative containing quinazoline moieties. The reaction of the H2L with copper(II) chloride in methanol resulted in the formation of a new methanol/water solvate containing discrete CuCl2L molecules. Cu(II) ion is coordinated by three nitrogen atoms (two from hydrazide and one from pyridine moieties) in a chelate fashion. Two chloride ions complete distorted square pyramidal coordination geometry. Unlike in H2L molecules, in CuCl2L the hydrazide N-atoms are deprotonated, and coordinated to Cu(II), while quinazoline N-atoms are protonated. In its crystal structure, CuCl2L molecules form dimeric motifs via OH⋯O, NH⋯O, OH⋯Cl hydrogen bond interactions. The adjacent dimers are stacked along the crystallographic b- and c-axis through NH⋯Cl and offset face-to-face π⋯π interactions. The FT-IR spectra analysis indicates a significant shift of CO stretching vibrations of uncoordinated carbonyl groups, that can be attributed to the electron-withdrawing effect of Cu(II) ion. The TG analysis indicates that the weakly bounded solvent molecules evaporate at lower temperatures and that the compound is stable up to 300 °C.

Replication elongates short DNA, reduces sequence bias and develops trimer structure.

The origin of molecular evolution required the replication of short oligonucleotides to form longer polymers. Prebiotically plausible oligonucleotide pools tend to contain more of some nucleobases than others. It has been unclear whether this initial bias persists and how it affects replication. To investigate this, we examined the evolution of 12-mer biased short DNA pools using an enzymatic model system. This allowed us to study the long timescales involved in evolution, since it is not yet possible with currently investigated prebiotic replication chemistries. Our analysis using next-generation sequencing from different time points revealed that the initial nucleotide bias of the pool disappeared in the elongated pool after isothermal replication. In contrast, the nucleotide composition at each position in the elongated sequences remained biased and varied with both position and initial bias. Furthermore, we observed the emergence of highly periodic dimer and trimer motifs in the rapidly elongated sequences. This shift in nucleotide composition and the emergence of structure through templated replication could help explain how biased prebiotic pools could undergo molecular evolution and lead to complex functional nucleic acids.

Mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN

HOIL-1L and SHARPIN are two essential regulatory subunits of the linear ubiquitin chain assembly complex (LUBAC), which is the only known E3 ligase complex generating linear ubiquitin chains. In addition to their LUBAC-dependent functions, HOIL-1L and SHARPIN alone play crucial roles in many LUBAC-independent cellular processes. Importantly, deficiency of HOIL-1L or SHARPIN leads to severe disorders in humans or mice. However, the mechanistic bases underlying the multi-functions of HOIL-1L and SHARPIN are still largely unknown. Here, we uncover that HOIL-1L and SHARPIN alone can form homo-dimers through their LTM motifs. We solve two crystal structures of the dimeric LTM motifs of HOIL-1L and SHARPIN, which not only elucidate the detailed molecular mechanism underpinning the dimer formations of HOIL-1L and SHARPIN, but also reveal a general mode shared by the LTM motifs of HOIL-1L and SHARPIN for forming homo-dimer or hetero-dimer. Furthermore, we elucidate that the polyglucosan body myopathy-associated HOIL-1L A18P mutation disturbs the structural folding of HOIL-1L LTM, and disrupts the dimer formation of HOIL-1L. In summary, our study provides mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN mediated by their LTM motifs, and expands our understandings of the multi-functions of HOIL-1L and SHARPIN as well as the etiology of relevant human disease caused by defective HOIL-1L.

O19 Differential pairing of transmembrane domain GxxxG dimerization motifs generates two HLA-DR conformers defined by selective binding by the mAb Tü36

O19 Differential pairing of transmembrane domain GxxxG dimerization motifs generates two HLA-DR conformers defined by selective binding by the mAb Tü36

Novel TCF4:TCF12 heterodimer inhibits glioblastoma growth.

TWIST1 (TW) is a pro-oncogenic basic helix-loop-helix (bHLH) transcription factor and promotes the hallmark features of malignancy (e.g., cell invasion, cancer cell stemness, and treatment resistance), which contribute to poor prognoses of glioblastoma (GBM). We previously reported that specific TW dimerization motifs regulate unique cellular phenotypes in GBM. For example, the TW:E12 heterodimer increases periostin (POSTN) expression and promotes cell invasion. TW dimer-specific transcriptional regulation requires binding to the regulatory E-box consensus sequences, but alternative bHLH dimers that balance TW dimer activity in regulating pro-oncogenic TW target genes are unknown. We leveraged the ENCODE DNase I hypersensitivity data to identify E-box sites and tethered TW:E12 and TW:TW proteins to validate dimer binding to E-boxes in vitro. Subsequently, TW knockdown revealed a novel TCF4:TCF12 bHLH dimer occupying the same TW E-box site that, when expressed as a tethered TCF4:TCF12 dimer, markedly repressed POSTN expression and extended animal survival. These observations support TCF4:TCF12 as a novel dimer with tumor-suppressor activity in GBM that functions in part through displacement of and/or competitive inhibition of pro-oncogenic TW dimers at E-box sites.